As an internal medicine resident, I developed an interest in lipoprotein metabolism and the pathogenesis of atherosclerotic disease. After a year of clinical of clinical training in Endocrinology and Metabolism at UCSF, I began basic research training in Dr. Stephen Young's laboratory at the Gladstone Institute. During the past three years, my research has focused on understanding several different aspects of apolipoprotein (Apo) B genetics and metabolism: 1) the effect of liver transplantation on apo B and apo E phenotype; 2) the expression of truncated apo B species in hepatoma cells; 3) the investigation of a unique allele causing hypobetalipoproteinemia, the apo B86 allele. The apo B86 allele, which contains a one nucleotide deletion (frameshift) in exon 26 of the apo B gene, results in the production of a truncated apo B species, apo B86. Remarkably, genetic and biochemical evidence from both affected family members and cell culture expression studies indicate that the apo B86 allele yields a full-length apo B100 in addition to apo B86. I have determined that the full-length protein is produced from the apo B 86 allele as a result of reading frame restoration, by a mechanism that appears to be unique in human genetics. The first specific aim of this project is to further define this mechanism for reading frame restoration. Site directed mutagenesis will be used to delineate the exact DNA sequence requirements for reading frame restoration. Mutant apo B constructs containing various DNA sequence changes in the region of the apo B86 mutation will be expressed in hepatoma cells. The apo B proteins (truncated vs. full-length) produced by the transformed cell lines will be examined by immunoblot. The mRNA from the transformed cell lines will be examined for evidence of an error in transcription that would restore the proper reading frame. Studies of the apo B86 protein suggest that apo B86 is incapable of associating with apo (a) to form lipoprotein (a) [Lp(a)]. It is widely assumed that apo B100 is linked to apo(a) by a disulfide linkage. Based on our results with apo B86, we hypothesize that one (or more of the carboxyl-terminal cysteines in apo B100 may be involved in forming a disulfide bridge with apo(a). The second specific aim is to use site directed mutagenesis and cell culture expression studies to evaluate the importance of the four carboxyl-terminal cysteines of apo B100 in its association with apo(a) to form Lp(a). Cell culture expression studies of apo B100 have obvious limitations with regard to investigating the role of apo B100 in the pathogenesis of atherosclerosis. Therefore, I plan to develop an animal model in which to test the hypothesis that overexpression of human apo B100 is atherogenic. The third specific aim will be to overexpress human apo B100 in transgenic mice. We plan to investigate the effects of overexpression of human apo B100 in transgenic mice on lipid levels and atherogenesis. In addition, we will develop an apo B86 transgenic mouse as a model in which to study reading frame restoration in the apo B86 allele, and we propose to use transgenic mice to develop allotype specific monoclonal antibodies to human apo B100.